1. Field of the Invention
The invention relates to the use of measurement of the polarization of the fluorescence emission from a labelled macromolecule in order to assess the binding of the labelled macromolecule to a second macromolecule. Thus, the invention is directed to analytic methods for determining such binding in a quantitative fashion. The invention is also directed to apparatus for conducting binding analyses using measurement of fluorescence polarization.
2. Description of the Related Art
The use of labeled oligonucleotides as probes in macromolecular analysis is an important technique in molecular biology. Oligonucleotides have been labeled with radioisotopes, enzymes or fluorescent molecules. Because of the relatively low molecular weights of oligonucleotides, and the common availability of instrumentation for their automated synthesis, oligonucleotides are often used in blot-hybridization procedures or in gel-retardation assays for the detection and qualitative evaluation of macromolecules with which they may associate. These macromolecules may be either proteins, RNA molecules or DNA molecules.
In a standard blot-hybridization procedure, the target macromolecule is separated by electrophoresis in a gel matrix, commonly agarose or polyacrylamide. It is then transferred to a membrane in such a way as to preserve its relative spatial positioning within the gel matrix and fix it stably to the membrane. Alternatively, the macromolecule may be attached to the membrane without prior electrophoresis. The presence of the macromolecule on the membrane is determined by binding to it a labeled oligonucleotide and subjecting the complex to autoradiography or, if the oligonucleotide is labeled with radioisotope, by scintillation counting.
In a standard gel retardation assay an oligonucleotide that has been labelled with radioisotope or other detectable moiety is electrophoresed in a gel matrix, commonly made of agarose or acrylamide, under non-denaturing conditions. The labelled oligonucleotide is also associated with a protein that may bind to the oligonucleotide and the mixture is electrophoresed on a gel, commonly in a neighboring lane, for comparing with the unassociated oligonucleotide. Because of its higher molecular weight and less negative charge, the protein will exhibit lower mobility in the gel than the unassociated oligonucleotide. If the oligonucleotide forms a stable complex with the protein, it will also exhibit a lower mobility than that of the unassociated oligonucleotide. Comparison of the mobility of the oligonucleotide mobility in the presence and absence of the protein allows qualitative determination of whether a complex forms between the two macromolecules. These basic methods are used for a very large variety of determinations in basic genetic research, genetic engineering, the medical sciences, and agricultural testing.
The present invention relates to a method for detecting and quantitating complexation between nucleic acids and proteins or other macromolecules which comprises the measurement of the polarization of fluorescence of an extrinsic fluorophore covalently coupled to an oligonucleotide. The oligonucleotide can be an oligodeoxyribonucleotide, an oligonribonucleotide or a copolymer of both. The nucleotide bases can be derivatized, as can the backbone chain. The oligonucleotide can be single-, double- or triple-stranded. The length of the oligonucleotide is determined by the specific experiment being conducted, but is preferably less than 40 residues long, more preferably being less than 25 residues long and most preferably 8-10 residues long.
The fluorophore is incorporated into the oligonucleotide at any position using standard automated DNA synthesis techniques and fluorescently labeled amino-linker compounds (e.g. those available from Clontech, Inc., La Jolla, Calif.). Alternately, unlabeled amino-linker compounds can be incorporated and subsequently labeled with the fluorescent compound. A variety of fluorophores may be used, including fluorescein, eosin, coumarin and dansyl derivatives. The fast rotation about the short axis of the oligonucleotide results in a low value of the fluorescence polarization of the probe covalently coupled to the oligonucleotide. This value is obtained by exciting the fluorescently labeled oligonucleotide with the appropriate wavelength of plane polarized exciting light and monitoring the emission of light polarized in the planes parallel and perpendicular to the plane of polarization of the exciting light. The fluorescence polarization is calculated as: EQU p=(I.sub.ll -I.sub.L)/(I.sub.ll +I.sub.L)
where I.sub.ll is the intensity of the emitted light polarized in a plane parallel to the plane of polarization of the exciting light and I.sub.L is the intensity of the emitted light polarized in a plane perpendicular to the plane of polarization of the exciting light. The anisotropy of the emission is EQU 2/3[1/(1p-1/3)]
This value is analogous to the polarization yet linear with respect to the total fluorescence emission intensity.
Fluorescence polarization is the basis for a series of patented assays for the clinical detection of drugs, steroids and other antigens (U.S. Pat. Nos. 4,269,511; 4,516,856; 4,585,862; 4,668,640; 4,784,961 4,902,630; 4,952,691; 5,066,426; European patent application 86102035.2). Although there are a number of variations, all of these assays rely on the observation of a large change in the fluorescence polarization of fluorescein upon changing its interaction with specific or non-specific antibodies when the drug of interest is present in the assay solution. If the presence of the drug results in the dissociation of the fluorescein-antibody complex, for example, then the fluorescence polarization will exhibit a large decrease. The sensitivity of these assays is less than 10 pM. Urios and Cittanova (15) describe the use of fluorescently labeled Fab fragments of antibodies to perform fluorescence polarization measurements. The sensitivity of their assay is 2.5 .mu.M. The use of a fluorescent-labeled oligonucleotide probe was reported by Murakami et al (17). Their reported sensitivity was 100 nanomolar. Giedroc et al. (16) used fluorescence anisotropy in evaluating the presence of single stranded DNA molecules. Their assays were conducted at oligonucleotide concentrations of 2 to 8 .mu.M.